Genetic and Biochemical Characterization of Halogenation and Drug Transportation Genes Encoded in the Albofungin Biosynthetic Gene Cluster.

Albofungin, a hexacyclic aromatic natural product, exhibits broad-spectrum antimicrobial activity. Its biosynthesis, regulation, and resistance remain elusive. Here, we report the albofungin (abf) biosynthetic gene cluster (BGC) from its producing strain Streptomyces tumemacerans JCM5050. The nascent abf BGC encodes 70 putative genes, including regulators, transporters, type II polyketide synthases (PKSs), oxidoreductase, and tailoring enzymes. To validate the intactness and functionality of the BGC, we developed an Escherichia coli-Streptomyces shuttle bacterial artificial chromosome system, whereby the abf BGC was integrated into the genome of a nonproducing host via heterologous conjugation, wherefrom albofungin can be produced, confirming that the BGC is in effect. We then delimited the boundaries of the BGC by means of in vitro CRISPR-Cas9 DNA editing, concluding a minimal but essential 60-kb abf BGC ranging from orfL to abf58. The orfA gene encoding a reduced flavin adenine dinucleotide (FADH2)-dependent halogenase was examined and is capable of transforming albofungin to halogen-substituted congeners in vivo and in vitro. The orfL gene encoding a transporter was examined in vivo. The presence/absence of orfA or orfL demonstrated that the MIC of albofungin is subject to alteration when an extracellular polysaccharide intercellular adhesin was formed. Despite that halogenation of albofungin somewhat increases binding affinity to transglycosylase (TGase), albofungin with/without a halogen substituent manifests similar in vitro antimicrobial activity. Halogenation, however, limits overall dissemination and effectiveness given a high secretion rate, weak membrane permeability, and high hydrophobicity of the resulting products, whereby the functions of orfA and orfL are correlated with drug detoxification/resistance for the first time. IMPORTANCE Albofungin, a natural product produced from Streptomycetes, exhibits bioactivities against bacteria, fungi, and tumor cells. The biosynthetic logic, regulations, and resistance of albofungin remain yet to be addressed. Herein, the minimal albofungin (abf) biosynthetic gene cluster (BGC) from the producing strain Streptomyces tumemacerans JCM5050 was precisely delimited using the Escherichia coli-Streptomyces shuttle bacterial artificial chromosome system, of which the gene essentiality was established in vivo and in vitro. Next, we characterized two genes orfA and orfL encoded in the abf BGC, which act as a reduced flavin adenine dinucleotide (FADH2)-dependent halogenase and an albofungin-congeners transporter, respectively. While each testing microorganism exhibited different sensitivities to albofungins, the MIC values of albofungins against testing strains with/without orfA and/or orfL were subject to considerable changes. Halogen-substituted albofungins mediated by OrfA manifested overall compromised dissemination and effectiveness, revealing for the first time that two functionally distinct proteins OrfA and OrfL are associated together, exerting a novel "belt and braces" mechanism in antimicrobial detoxification/resistance.

Mode of action of antimicrobial agents albofungins in eradicating penicillin- and cephalosporin-resistant Vibrio parahaemolyticus biofilm.

Albofungin is a promising broad-spectrum antimicrobial compound against multidrug-resistant bacteria. In the present study, we further investigated albofungin's biofilm eradication activity and its potential mode of action against drug-resistant Vibrio parahaemolyticus. Among all derivatives, albofungin exhibited the best antibiofilm and antibacterial activity with rapid killing effects at 0.12 µg mL-1. Confocal microscopy observation exhibited that albofungin disrupted V. parahaemolyticus biofilms by killing or dispersing biofilm cells. Meanwhile, scanning electron microscope and fluorescent staining experiments demonstrated that albofungin rapidly destroyed the integrity and permeability of the bacterial cell membrane. Moreover, this study revealed an antibiofilm mechanism of albofungin involving inhibition of peptidoglycan biosynthesis, flagella assembly pathways, and secretion system proteins in V. parahaemolyticus by quantitative proteomics and validation experiments. Our results highlighted albofungin's mechanism of action in planktonic cells and biofilms and suggested further development and potential applications of albofungin for treating infections caused by penicillins-and-cephalosporins-resistant V. parahaemolyticus. IMPORTANCE Infections caused by multidrug-resistant bacteria, as well as a scarcity of new antibiotics, have become a major health threat worldwide. To tackle the demand for new and effective treatments, we investigated the mechanism of action of albofungin, a natural product derived from Streptomyces, which exhibits potent antimicrobial activity against multidrug-resistant bacteria. Albofungin showed potent biofilm eradication activity against penicillins-and-cephalosporins-resistant Vibrio parahaemolyticus, which expresses a novel metallo-ß-lactamase and, thus, reduces their sensitivity to various antibiotics. We observed membrane disruption and permeation mechanisms in planktonic cells and biofilms after albofungin treatment, while albofungin had a weak interaction with bacterial DNA. Moreover, the antibiofilm mechanism of albofungin included inhibition of peptidoglycan biosynthesis, flagellar assembly pathways, and secretion system proteins. Our finding suggested potential applications of albofungin as an antibacterial and antibiofilm therapeutic agent.

Discovery, Yield Improvement, and Application in Marine Coatings of Potent Antifouling Compounds Albofungins Targeting Multiple Fouling Organisms.

Marine biofouling caused huge economic losses of maritime industries. We aim to develop high-efficient, less-toxic, and cost-effective antifoulants to solve the problems of biofouling. In this study, we described the antifouling compounds albofungin and its derivatives (albofungin A, chrestoxanthone A, and chloroalbofungin) isolated from the metabolites of bacterium Streptomyces chrestomyceticus BCC 24770, the construction of high-yield strains for albofungin production, and application of albofungin-based antifouling coatings. Results showed that these albofungins have potent antibiofilm activities against Gram-positive and Gram-negative bacteria and anti-macrofouling activities against larval settlement of major fouling organisms with low cytotoxicity. With the best antifouling activity and highest yield in bacterial culture, albofungin was subsequently incorporated with hydrolyzable and degradable copolymer to form antifouling coatings, which altered biofilm structures and prevented the settlement of macrofouling organisms in marine environments. Our results suggested that albofungins were promising antifouling compounds with potential application in marine environments.

Discovery, Bioactivity Evaluation, Biosynthetic Gene Cluster Identification, and Heterologous Expression of Novel Albofungin Derivatives.

The crude extract of Streptomyces chrestomyceticus exhibited strong and broad activities against most "ESKAPE pathogens." We conducted a comprehensive chemical investigation for secondary metabolites from the S. chrestomyceticus strain and identified two novel albofungin (alb) derivatives, i.e., albofungins A (1) and B (2), along with two known compounds, i.e., albofungin (3) and chloroalbofungin (4). The chemical structures of the novel compounds were elucidated using HRMS, 1D and 2D NMR, and electronic circular dichroism spectroscopy. The draft genome of S. chrestomyceticus was sequenced, and a 72 kb albofungin (alb) gene cluster with 72 open reading frames encoding type II polyketide synthases (PKSs), regulators, and transporters, and tailoring enzymes were identified using bioinformatics analysis. The alb gene cluster was confirmed using the heterologous expression in Streptomyces coelicolor, which successfully produced the compounds 3 and 4. Furthermore, compounds 1-4 displayed remarkable activities against Gram-positive bacteria and antitumor activities toward various cancer cells. Notably, compounds 1 and 3 showed potent activities against Gram-negative pathogenic bacteria. The terminal deoxynucleotidyl transferase (dUTP) nick-end labeling and flow cytometry analysis verified that compound 1 inhibited cancer cell proliferation by inducing cellular apoptosis. These results indicated that albofungins might be potential candidates for the development of antibiotics and antitumor drugs.

Albofungin and chloroalbofungin: antibiotic crystals with 2D but not 3D isostructurality.

The potent antibiotics albofungin [systematic name: (1S,4R,8aR)-13-amino-1,15,16-trihydroxy-4-methoxy-12-methyl-3,4,8a,13-tetrahydro-1H-xantheno[4',3',2':4,5][1,3]benzodioxino[7,6-g]isoquinoline-14,17(2H,9H)-dione, C27H24N2O9, 1] and its chlorinated analogue chloroalbofungin (the 11-chloro analogue, C27H23ClN2O9, 2) have been crystallized following their isolation from the bacterial strain Streptomyces chrestomyceticus and their structures determined by single-crystal X-ray diffraction. The novel N-aminoquinolone molecular arrangement shows N-N bond lengths of 1.4202 (16) and 1.424 (2) Šin 1 and 2, respectively. The regiochemistry of chloro substitution in the A-ring is para to the quinolone O atom, with a C-Cl bond length of 1.741 (2) Å. The absolute stereochemistry at three chiral centres of the xanthone rings (i.e. 10S, 13R and 19R) is confirmed. Both compounds crystallize in chiral Sohncke space groups consistent with enantiopurity, but are not fully isostructural. A preserved supramolecular construct (SC) confers two-dimensional (2D) isostructurality, but the SC self-associates via either a twofold screw operation in 1, giving a monoclinic P21 structure, or a twofold rotation in 2, affording a monoclinic C2 structure with a doubled unit-cell axis.